Lactobacillus paracasei TKU012 所生產蛋白酶特性研究及其應用

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Title:	Lactobacillus paracasei TKU012 所生產蛋白酶特性研究及其應用
Other Titles:	Characterization of a protease from lactobacillus paracasei TKU012 and its application
Authors:	王俊元;Wang, Chun-yuan
Contributors:	淡江大學生命科學研究所碩士班王三郎;Wang, San-lang
Keywords:	Lactobacillus paracasei;耐酸耐膽鹽;Lactobacillus paracasei;protease;acid and bile resistant
Date:	2007
Issue Date:	2010-01-11 02:26:17 (UTC+8)
Abstract:	益生菌用於發酵在各種食物已有多年歷史。近年來許多研究指出，藉由益生菌(probiotics)的攝取可平衡腸道菌相並有助於維持身體健康。這項研究的目的是以研究Lactobacillus sp. TKU 012所生產之蛋白酶的純化和定性及其對酸和膽汁耐受力及其應用。由嬰兒吐奶中所篩選出之菌株Lactobacillus sp.TKU012能以烏賊軟骨粉末作為生產蛋白酶之主要營養源。Lactobacillus sp.TKU012生產蛋白酶之較適培養條件為: 將含有1%烏賊軟骨粉末、0.1%K2HPO4、0.05%MgSO4．7H2O之100mL液體培養基(pH9)於250mL之三角錐形中，於25℃振盪培養3天後，可得最大蛋白酶活性。利用Lactobacillus sp.TKU012之蛋白質酶較適生產條件大量培養，所得發酵液經硫酸銨沉澱、DEAE-Sepharose離子交換層析及Sephacryl S-100膠體層析一連串分離步驟，可純化出一種蛋白質酶。TKU012 蛋白酶之生化特性經分析結果，其最適反應溫度50℃、最適反應pH10、熱安定性為<60℃、pH安定性5-10及利用SDS-PAGE分析出來的分子量約為49kDa;活性會受到PMSF的抑制，屬於絲胺型的蛋白質酶;其活性會受到Cu2+、Ba2+ 和Zn2+ (5mM)的抑制，蛋白質酶的殘餘活性分別只剩53%、44%與32%;且純化後的酵素在0.5(mM)SDS，0.5%(v/v)Tween 40，0.5%(v/v)Triton X-100界面活性劑存在下殘餘活性分別為98%、67%及45%。有機溶劑對酵素活性及安定性影響方面，在甲苯、乙醚、與丙酮(25%, v/v)等有機溶劑下，蛋白質酶仍有50%以上的殘餘活性; 而將酵素與有機溶劑(20%, v/v)於4℃與25℃下，待反應10天後，皆仍然保有75%以上的殘餘活性。在其它研究，TKU 012在對酸鹼度2.5 磷酸鹽緩衝的這個測試之耐受性上，它在酸鹼度2.5時可達到103 cfu/ml。耐膽鹽性試驗方面，在含0.3%牛膽鹽之MRS plus broth 中培養24 小時存活率達90％以上。經過耐酸性(pH2.5)和耐膽鹽性(0.3%)測試，TKU 012模擬消化過程之測試及TKU 012菌株的膽鹽耐受性可達到80%以上。 Probiotics have been used in various fermented foods for many years. Resent reports indicated that digest probiotics are microbial food supplements, which beneficially affect the balance of intestinal microflora and host’s healthy. The purpose of this study was to investigate the purification and characteristics of a protease from Lactobacillus paracasei TKU012and its acid and bile resistant ability discussion . The protease-producing strain, Lactobacillus paracasei TKU012, was isolated from infant Spits milk . The optimized culture medium was composed of 1% squid pen powder(SPP), 0.1%K2HPO4, 0.05%MgSO4．7H2O at pH9. The strain was incubated in 100mL of above liquid medium and kept shaking at 25℃ for 3 days. The TKU012 protease was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose column chromatography and Sephacryl S-100 gel chromatography. The molecular mass of TKU012 protease determined by SDS-PAGE was approximately 49kDa.The optimum temperature, optimum pH , pH stability and thermal stability of TKU012 protease was 50℃, pH 10, pH5-10 and<60℃ . The protease was characterized as a Serine protease because it was inactivated by PMSF. Additionally,the pure protease retained 98% ,67% ,and 45% of its original activity in the presence of 0.5(mM)SDS, 0.5%Tween 40 and 0.5%Triton X-100, respectively. In the presence of organic solvent such as toluene, ethyl ether and acetone, the protease retained more than 50% of its activity. The TKU012 protease retain over 75 % of its activity by pre-incubation in the organic solvent at 4℃ and 25℃ for 10 days. Additionally, TKU012 showed tolerance properties in this test on pH 2.5 phosphate buffer, it could achieve 103 cfu/ml . In the bile resistance test, it viability can achieve higher than 90% survival population in MRS plus broth (include 0.3% bull bile salt) after being incubated for 24hrs.According to the acid and bile resistance test, TKU012 take the imitating digestive tract test and it was higher than 80% survival population.
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